I'm not sure what a "TOP10 transformant" is but assume it must be an E. coli K12
strain.
Thiamine (B1) is very stable in my hands, whether as solid thiamine
hydrochloride stored at room temp. or as a 10 mg/ml stock stored at 4 degrees.
Crystals of the hydrochloride salt are very soluble in water. I do not add NaOH.
I filter sterilize the stock solution, but thiamine is reputedly stable to
autoclaving. I use it at a final concentration of 5 ug/ml which is probably in
excess of the need of E. coli thi mutants. Are you sure that you are not using
thymine by mistake? I have seen that.
For lysine, I use a 10 mg/ml stock with a final of 50 ug/ml. Again, I filter
sterilize. Be sure not to use D-Lysine by mistake.
Growing your strain in M9 supplemented with LB would confirm that the problem is
with your supplements, not the M9 basal medium.
Emir Khatipov wrote:
> Hi,
> I have problems with growing TOP10 transformants in M9 minimal medium.
>> Could someone experienced in using M9 minimal medium advise me what is the
> most appropriate way of preparing and handling/storing thiamine solution.
> What should be the concentration of the stock solution so that thiamine will
> dissolve readily in water, and whether it should be prepared fresh or can be
> stored, how long? I was advised to add up to 10ug/ml thiamine to the medium,
> and I make 1000 x water solution by mixing thiamine crystals with water and
> adding alkali.
>> The TOP10 cells I am using cells that are L-lysine auxotrophs, so any
> additional comments on lysine solution would be appreciated, too (making,
> storing).
>> I make the M9 medium according to conventional protocols. Since there is
> nothing special about M9 salts and glucose, I don't think I am screwing
> something up there. I guess my cells do not grow because I do something
> wrong when making thiamine or lysine.
>> I know thiamine is light-sensitive and I store it in the dark. Anyway, I
> could not get my cells grow even with freshly made thiamine solution.
>> I also know that I could add up to ~4% LB to facilitate growth, but I do not
> want to do that.
>> Thanks.
> Emir